Transgenic mouse expressing human lipoprotein (a) with disabled vitamin c gene and its use as a disease treatment model

ABSTRACT

The invention discloses novel model of transgenic mammal, a method of crossbreeding transgenic mammal and the use of the transgenic mammal for assessing prevention and/or treatment methods for cardiovascular and other diseases related to lipoprotein(a). The transgenic mammal expresses human apolipoprotein (a) (apo(a)) and human apolipoprotein B-100 (apo B-100) genes and produces human lipoprotein (a), apo (a) and apo B-100 and produces no vitamin C. This novel dual transgenic mammal is the ideal model for testing pharmaceutical compounds for efficacy and usefulness in the prevention and/or treatment of human diseases.

CROSS REFERENCE TO RELATED APPLICATION

This instant application is a continuation of U.S. Utility applicationSer. No. 14/025,532 filed on Sep. 12, 2013. The pending and now allowedU.S. Utility application Ser. No. 14/025,532 is hereby incorporated byreference in its entireties for all of its teachings.

FIELD OF TECHNOLOGY

This disclosure relates generally to a transgenic mouse that has beengenetically altered to express human lipoprotein (a) and a disabled genefor the expression of Vitamin C. More specifically the two defininghuman proteins of lipoprotein (a), apolipoprotein (a) and apolipoproteinB-100 gene may be expressed either individually or in combinationwithout the expression of Vitamin C gene. This application containssequence listing that has been submitted as an ASCII file namedRIPLLC018017US1sequence_ST25, the date of creation Sep. 12, 2013, andthe size of the ASCII text file in bytes is 2 kb. The dual transgenicmouse embryo referred to as Rath M Human Lipoprotein(a);Gulo(−/−) hasthe Jackson Stock# 912329, having been deposited in The JacksonLaboratory on Apr. 8, 2013.

BACKGROUND OF THE INVENTION

Cardiovascular disease is responsible for half of the deaths in theindustrial world. Over the past decades a new risk factor for thisdisease has emerged, lipoprotein (a)(Lp (a)). Lp (a) is has been shownto be an independent risk factor for myocardial infarctions, (Rhoads G Get. al., 1986, Clarke et al, 2009) cerebrovascular disease (Zenker G et.al.,1986) and other forms of cardiovascular disease. Furthermore, Lp(a)has been identified as a significant component of human atheroscleroticplaques (Rath M et. al., 1989). Aside from Niacin, there is currently noaccepted effective treatment available in clinical cardiology to lowerLp(a) plasma levels or to prevent its deposition inside the vascularwall.

Lp(a) was discovered by Kare Berg in 1963 (Berg, K et. al., 1963). It iscomposed of a low-density-lipoprotein molecule (LDL) and apolipoprotein(a) (apo(a)), a glycoprotein attached to the structural protein of LDL,apolipoprotein B-100 (apo B), via disulfide bonds. The cDNA of apo(a)shows a strong homology with plasminogen containing multiple repeats ofplasminogen kringle IV. Due to this homology apo(a) binds tofibrinogen/fibrin and attenuates fibrinolysis (McLean, J W et. al.1987).

Lp(a) is primarily found in humans and subhuman primates and theappearance of the apo(a) gene was dated to about 40 million years ago,about the time of the divergence of the Old World and New World monkeys(McLean, et al. Nature, 1987). This was also the time point duringevolution when the ancestor of man lost the ability for endogenousascorbate synthesis due to a mutation in the gene encoding forgulonolactone oxidase (GULO), an essential enzyme for the conversion ofglucose to ascorbate (vitamin C) (Chatterjee I B, 1973, Nikishimi M etal., 1991).

The significance of ascorbate deficiency in initiating the process ofatherogenesis has recently been documented in mouse unable to expressthe gene for L-gulonolactone oxidase (GULO−/−) (Maeda N et. al., 2000).

Roy et. al. (2003, U.S. Pat. No. 6,512,161) discusses several failedattempts to create animal models for expressing specifically Lp(a) inmodels such as rats, mouse and guinea pigs and state that they don'talways represent human metabolism and human-related diseases. In theirstudy they invented a rabbit model expressing human apo (a) and humanapo B-100 genes. However, the transgenic rabbit developed by Roy et. al.(2003) also does not mimic the human physiology with respect to anotherkey metabolic aspect: unlike humans, rabbits are able to produce theirown Vitamin C.

There exists a need for a dual transgenic mammal model displaying theseunique genetic features in order to develop new preventive andtherapeutic approaches related to them.

SUMMARY

The current application discloses a method of making and using a dualtransgenic mammal (mouse, rat and other mammalian species) thatpossesses the genes for human apo (a) and/or apo B-100 and produceshuman (Lp(a) while, at the same time, being unable to produce vitamin C.In one embodiment, a third strain of transgenic mammal was obtained bycrossbreeding the first knockout strain mammal and second strain oftransgenic mammal (may be a mouse or other animals) expressing a set ofgenes that are human in nature, wherein the first strain is a knockoutmammal possessing a non-functional L-gulonolactone oxidase (GULO)(GULO−/−) and hence produces no Vitamin C, wherein the second strain ofmice expresses human apo (a) gene (apo (a)+) and produces apolipoprotein(a)(apo (a)), wherein the third strain of transgenic mice possessesnon-functional L-gulonolactone oxidase (GULO−/−) gene and a functionalhuman apo (a) gene (apo (a)+). Hence this third strain of mammal willnot produce vitamin C but will produce human apo (a).

In another embodiment, a fifth strain of transgenic mammal was made bycrossbreeding the first knockout strain mammal possessing non-functionalL-gulonolactone oxidase (GULO−/−) gene and the fourth strain expressinghuman apo B-100 gene (apo (B-100)+), wherein the fifth strain of mammalpossesses non-functional L-gulonolactone oxidase (GULO−/−) gene and afunctional human apo B-100 gene (apo (B-100)+). Hence the fifth strainof transgenic mammal will not produce vitamin C and will produce humanapolipoprotein B-100.

In another embodiment, third strain and fifth strain of transgenicmammal were crossbred to obtain a novel dual transgenic mammal (may be amouse for example) that had a knockout GULO gene (GULO−/−), a functionalhuman apo B-100 gene (apo(B-100)+) and a functional human apo (a) gene(apo (a)+). The novel dual transgenic mammal will hence, produceapolipoprotein (a)(apo (a)) and/or apolipoprotein B-100 (apo B-100) aswell as the complete lipoprotein(a) particle (Lp(a) and will not producevitamin C. This novel double transgenic mammal model resembles the humansystem with respect to the inability of endogenous ascorbate synthesisand, congruently, the expression of apo(a), apo (B-100) as well as thecomplete lipoprotein(a) particle (Lp(a)). In the instant disclosure amouse model is used but other mammals may be used and crossbreeding,insertion of genes or deletion of genes may be done to produce thesedual transgenic mammals to express or suppress human genes in anycombination.

In one embodiment, a mammal, a mouse whose genome lacks the ability forendogenous ascorbate synthesis and—simultaneously—expresses a human apo(a) is disclosed. In another embodiment, a dual transgenic mammal, amouse, whose genome lacks the ability for endogenous ascorbate synthesisand—simultaneously—expresses a human apo B-100 is disclosed. In anotherembodiment, a transgenic mammal, a mouse, whose genome lacks the abilityfor endogenous ascorbate synthesis and—simultaneously—produces a humanLp(a) is disclosed. An animal model may be created by crossbreeding,gene insertion or other methods of molecular biology and/or geneticengineering.

In one embodiment, the novel dual transgenic mouse to be used as a modelfor cardio vascular disease (CVD) study. In another embodiment, the dualtransgenic mice to be used as a model for treating CVD like diseases. Inanother embodiment, the dual transgenic mouse may be used as a model totest new and old drugs to treat diseases associated with Lp(a) synthesisand lack of vitamin C production.

In one embodiment, the dual transgenic mouse model may be used fortesting effect of various drugs involved in ischemic heart disease,cardiovascular diseases, including coronary artery disease,cerebrovascular disease (stroke), renal vascular disease, peripheralvascular disease, aneurysms, thrombotic conditions, other forms ofvascular disease, inflammatory conditions, as well as infectiousdiseases, neuroinflammatory and neurodegenerative diseases.

In one embodiment, a process for making a dual transgenic mammal whichlacks the ability for endogenous ascorbate synthesisand—simultaneously—is capable of producing human apolipoprotein(a) orhuman apolipoprotein B or Lp(a) comprising mating a first mammal inwhich the ability for ascorbate synthesis has been genetically deletedwith a second mammal which has a genome encoding human apo (a) or humanapo B-100 or both of these apolipoproteins in such a manner that theycombine in vivo in said transgenic mammal to produce the complete humanlipoprotein(a) particle (Lp(a)).

In another embodiment, a method for determining whether a compound cantreat atherosclerosis or an undesirable plasma lipid profile comprising:a) comparing the lipid profile or state of atherosclerosis in a firsttransgenic mammal fed a diet generally known to be atherogenic andtreated with said compound, to the lipid profile or state ofatherosclerosis in a second transgenic mammal fed the same atherogenicdiet but not treated with said compound; and determining the potentialtherapeutic effect of said compound based upon comparative evaluation ofthe lipid profile or state of atherosclerosis in said first and secondtransgenic mammal; wherein said first and second transgenic mammal eachbeing a transgenic mammal.

In one embodiment, a treatment method wherein the drug to treat theeffect of high Lp(a) in the absence or in presence of micronutrientssuch as vitamin C is observed using transgenic mouse that does notproduce vitamin C and produces human Lp(a).

The composition, method, and treatment disclosed herein may beimplemented in any means for achieving various aspects, and may beexecuted in a form suitable for the mammal.

BRIEF DESCRIPTION OF DRAWINGS

Example embodiments are illustrated by way of example and not limitationin the Figures of the accompanying drawings, in which like referencesindicate similar elements and in which:

FIG. 1 shows the scheme used for creating the dual transgenic mouse.

FIG. 2 shows the expression of disulfide linked human apo(a) and humanapoB-100 as assembled Lp(a) with a lipid globule in male and femaletransgenic mouse with both human apo(a) and human apo B-100 genes, butnot in male and female transgenic mouse without both genes.

FIG. 3 shows the expression of human apo(a) protein in female and malemice.

FIG. 4 shows the expression of human apo B-100 protein in female andmale transgenic mouse in LDL particles and Lp(a) particles.

FIG. 5 shows the serum ascorbate level in transgenic female and malemouse (26-29 weeks old) supplemented with Vitamin C or deprived ofVitamin C in micromoles/liter (uM).

FIG. 6 shows Serum Protein Immunofixation Electrophoresis (SPIFE)cholesterol profile of hypoascorbemic or fully ascorbate-supplementedLp(a)+GULO (−/−) mouse.

FIG. 7 shows Immunofixation Electrophoresis (IFE) apo(a)-particleprofile of hypoascorbemic or fully ascorbate-supplemented Lp(a)+GULO(−/−) mouse.

FIG. 8 shows IFE Human Apo B-Particle profile of hypoascorbemic or fullyascorbate-supplemented Lp(a)+GULO (−/−) mouse.

Other features of the present embodiments will be apparent from theaccompanying drawings and from the detailed description that follows.

DETAILED DESCRIPTION

The invention discloses novel dual transgenic mammal/mouse, method ofcrossbreeding a dual transgenic mammal (may be a mouse or other animals)and the use of the dual transgenic mammal/mouse for assessing treatmentmethod for cardiovascular and related diseases. The dual transgenicmouse expresses human apolipoprotein (a) and apolipoprotein B-100 genesand produces apolipoprotein (a) and apolipoprotein B-100 as well ascomplete human lipoprotein (a) particles in this mouse which,simultaneously, does not express L-gulonolactone oxidase (GULO −/−) and,consequently, does not produce vitamin C. This novel dual transgenicmouse is the ideal model for testing pharmaceutical compounds fortreatment efficacy and usefulness for Lp(a) modulation with a variety ofbiological and/or pharmaceutical compounds, including but not limitedto, nutrition, pharmaceutical drugs and treatment methods that affecthuman beings.

Although the present embodiments have been described with reference tospecific example embodiments, it will be evident that variousmodifications and changes may be made to these embodiments withoutdeparting from the broader spirit and scope of the various embodiments.

Cross Breeding of Mammal/Mouse:

An animal model may be created by crossbreeding, gene insertion or othermethods of molecular biology and/or genetic engineering. In an exemplaryexample crossbreeding of a knock mouse and a specific human expressinggene containing mouse is disclosed to create a dual transgenic mouse.

BALB/cBy-Gulo (−/−) mouse: The strain, BALB/cBy-Gulo^(sfx)/J was aspontaneous mutation, mapped to the Gulonolactone oxidase locus, thegene for vitamin C synthesis. The GULO (−/−) strain mouse (the firstknockout strain of transgenic mouse) was generated from heterozygous(hemizygous) GULO (+/−) breeders obtained from The Jackson Laboratory(Table 1). The mouse was bred under vitamin C supplementation until anadequate number of homozygous GULO (−/−) breeders were obtained.

TABLE 1 GULO (−/−) strain description: Allele Symbol Gulo^(sfx) AlleleName spontaneous fracture Allele Type Spontaneous Strain of OriginBALB/cBy-Rasa3 Gene Symbol and Name Gulo, gulonolactone (L-) oxidaseChromosome 14 Gene Common Name(s) AU018375; BC028822; L-gulono-gamma-lactone oxidase; MGC: 29968; MGC: 37793; MGC: 37880; cDNA sequenceBC028822; expressed sequence AU018375; sfx; spontaneous fracture;General Note This spontaneous mutation appeared in a BALB/cBy-scatcolony at The Jackson Laboratory. The scat and sfx mutations wereseparated from each other by backcrossing BALB/cBy-scat mouse toBALB/cBy mouse and observing F2 offspring for those that exhibited thesfx phenotype but not the scat phenotype. Molecular Note The mutation inthe sfx mouse is a deletion that includes the entire Gulo gene. [MGI RefID J: 95128]

Maintenance of GULO (−/−) mouse: GULO (−/−) mouse are unable tosynthesize their own vitamin C; therefore this nutrient needs to bepresent in the mouse diet. Vitamin C was provided in a double distilleddrinking water containing 150 mg/L ascorbic acid (Sigma) and 0.01 mMEDTA (Sigma) to stabilize vitamin C from degradation by interaction withtrace metals. The water also contained 10 g/L of sucrose in order tomask a taste offensive to mouse. Water was changed twice a week. Inaddition, these mouse received food fortified with 500 ppm Lascorbyl-polyphosphate, the standard veterinary feed source of stablevitamin C milled at Test Diet as Modified Custom Lab Diet #5A38.

Human Apo (a) Mouse:

The Human apo(a) mouse (second strain of transgenic mouse) was obtainedfrom the Mutant Mouse Regional Resource Centers (MMRRC), supported bythe NIH. The strain, FVB/N-Tg(LPA, LPAL2, PLG)1Hgc/Mmmh, was createdusing a 270 kb YAC that harbors human apo(a) and apo(a) like andplasminogen genes. The donor was Edward M. Rubin, M.D., Ph.D., LawrenceBerkeley National Laboratory. Founder mice were bred until sufficientnumber of apo(a)+Gulo wildtype mice were obtained for crossbreeding.Genotyping for the transmission and presence of the transgene wasperformed at Transnetyx (Cordova, TN) upon tail clip tissue andtransgenic mutants confirmed positive for apo(a) in the genome selectedfor cross-breeding.

Human Apo B-100 Mouse:

The human apoB-100 mouse (fourth strain of transgenic mammal/mouse) wasobtained from Taconic Farms, Inc. under academic research agreement. Thestrain, B6.SJL-Tg(APOB)1102Sgy N20+?, or apoB-100 mouse, was developedby MacRae F. Linton et. al. of the Gladstone Institute of CardiovascularDisease by microinjecting the human apolipoprotein B100 gene intoC57BL/6J×SJL zygotes. The resultant mouse was backcrossed to C57BL/6 for4 generations (N4). Taconic received stock from Xenogen Biosciences inMay 1996. The mouse was maintained by backcrossing hemizygous Apo(B-100) mouse with C57BL/6NTac inbred mouse. Hemizygous mouse were bredto obtain homozygous Apo (B-100) mouse. Genotyping for transmission andpresence of the transgene Human Apo (B-100) in the genome was performedat Transnetyx upon tail clip tissue and transgenic mutants selected forcross-breeding.

Cross Breeding Steps Leading to Generating a Mouse Strain ProducingHuman Lp(a):

FIG. 1 shows the various steps used for crossbreeding several strains ofmouse to obtain a dual transgenic mouse. The terms mouse and mice areused interchangeably and they all mean mouse in the instantspecification. Crossbreeding (108) of human apo (a) mice (104) (thesecond strain of mammal)+GULO (−/−) (102) (a first knockout strainmammal) mice/mouse to produce a third strain of transgenic mammal/mousethat expresses the human apolipoprotein (a) gene (apo (a)+) and,simultaneously lacks the GULO gene (GULO−/−) (112) was performed. A GULO(−/−) (102) (a first knockout strain mammal) and human apo B-100 mouserepresented as fourth strain of mammal (106) was crossbred (108) toproduce a fifth strain of mouse that the fifth strain of mammal lacksthe GULO gene (GULO−/−) and expresses the human apolipoprotein B-100gene (apo (B-100)+) (114). Two transgenic founding strains were thusobtained for further crossbreeding:

-   -   Human apo(a)+GULO (−/−) mouse—third strain of transgenic mouse    -   Human apo B-100+GULO (−/−)mouse—fifth strain of transgenic        mouse.

Crossbreeding (108) the Founding Strains for Obtaining MouseStrain:Human Lp(a)+GULO (−/−) (116):

Newly generated mouse breeders of human apo(a)+GULO (−/−) mouse (112)and human apo B100+GULO (−/−) (114) were subsequently crossed (108) withone another to generate the new mouse strain: human Lp(a)+GULO (−/−)mice (116) which had human apo(a)+human ApoB-100+GULO (−/−), named as“Rath M Human Lipoprotein(a);Gulo (−/−)” strain (116). The dualtransgenic mouse embryo referred to as Rath M HumanLipoprotein(a);Gulo(−/−) has the Jackson Stock# 912329, having beendeposited in The Jackson Laboratory on Apr. 8, 2013.

Genotyping:

Genotyping for the GULO locus and its homozygosity was performed viaTaqman FAM Probe Real Time-PCR at Transnetyx upon tail clip tissuederived DNA obtained using standard DNA isolation and PCR techniques.Transgene presence for human apo B-100 and human apo(a) were alsoconducted at Transnetyx.

Tail clips were obtained from mouse under anesthesia and then shipped toTransnetyx where the following probe sets were designed and used forReal-Time PCR detection of genomic DNA presence or absence (primers usedare shown below in the Tables 2, 3 and 4. Litters were genotyped viatail clip Taqman PCR at Transnetyx and those positive for genomictransgenes apo(a) and apoB-100, as well as homozygous knockout mutationfor the L-gulonolactone-oxidase gene, GULO (−/−), which indicatesvitamin C synthesis defect, were selected and labeled as “Rath M HumanLipoprotein(a); Gulo (−/−)” founder mouse. In FIG. 2 we show that thedisulfide linked lipoprotein (a) is formed in the dual transgenic mouseand shown as Lp(a) GULOKO F1, F2, M1 and M2. Similarly, the lack ofLp(a) in apo(a)+mouse without human apoB-100 expression and apo B-100+mice without apo(a) expression is also shown.

TABLE 2 GULO testing: Gulo-1 KO CTAGTGTAGTCTAGGTGATAAGGATCAACT—Seq 1Forward Primer: Gulo-1 KO CAGCTCAGAGAGAGAATGAATCACA—Seq 2 ReversePrimer: Reporter 1: CTGACATCCCTTAGGAGTTC—Seq 3 Gulo-1 WTAGATGTGTTCCAGGCTGCAA—Seq 4 Forward Primer: Gulo-1 WTCACACACTGCAGGGTGACA—Seq 5 Reverse Primer: Reporter 1:CTGCCTGGGTGTTATC—Seq 6

Genotype Results Interpretation: Gulo-1 KO+, Gulo-1 WT+=HemizygousVitamin C generating mouse. Gulo-1 KO−, Gulo-1 WT+=Homozygous wild typeVitamin C generating mouse. Gulo-1 KO+, Gulo-1 WT−=Homozygous Vitamin Cdefective mouse.

Mouse homozygous for the knockout, GULO(−/−) mouse was selected forcross-breeding.

TABLE 3 B: Human apo(a) testing HuLPA-1 Tg (HumanCACTACATTTTGTGCCAGAGATGGA—Seq 7 apo(a) transgene) Forward Primer:HuLPA-1 Tg CCCTGTCCTGAGGCTCCTTA—Seq 8 Reverse Primer: Reporter 1:TCAGCAGCCCTCTTCC—Seq 9

Genotype Results Interpretation: +=Human apo(a) gene positive. −=Humanapo(a) gene negative. Mouse positive for the transgene were selected forcross-breeding.

TABLE 4 Human apo B-100 testing primers ApoB Tg (HumanAGGTTTAACTCCTCCTACCTCCAA—Seq 10 ApoB100 Transgene) Forward Primer: ApoBTg TGAGGGAGAGGGTTCCATCTT—Seq 11 Reverse Primer: Reporter 1:ACCAGATAACAGGAAGATATG—Seq 12

Genotype Results Interpretation: +=Human apoB-100 gene positive. −=HumanapoB-100 gene negative. Mouse positive for the transgene were selectedfor cross-breeding.

The genotype of the Lp(a); GULO (−/−) mouse is denoted as h apo(a)+; hapoB-100+; GULO(−/−). The mouse must continually be maintained onvitamin C supplementation as described above.

Confirming the transgene mouse generation at the level of protein: Thepresence of human apo(a) and human apo B-100 proteins in mouse sera wasdetermined by ELISA in the serum drawn from the GULO (−/−) mouse, theapo(a)+GULO(−/−) mouse, the apoB+GULO (−/−) mouse, and the Lp(a)+GULO(−/−) mouse.

TABLE 5 Lp(a); GULOKO mice and hApoB; GULOKO mice express human ApoB viaAssayPro ApoB ELISA Sample name ug/mL ug/ml * 20000 mg/dL gko m1 0 gkom2 0.001153 gko f1 0.000374 gko f2 −0.00036 Lp(a); gko m1 0.014717294.3472224 29.4 mg/dL Lp(a); gko m2 0.015867 317.346142 31.7 mg/dLLp(a); gko f1 0.041693 833.8570197 83.3 mg/dL Lp(a); gko f2 0.041411828.2183041 82.8 mg/dL apo(a); gko m1 0.000185 apo(a); gko m1 −0.00018apo(a); gko f1 −0.00054 apo(a); gko f2 −0.00018 hApoB; gko m1 0.0568421136.847637  114 mg/dL hApoB; gko m2 0.020853 417.0655494 41.7 mg/dLhApoB; gko f1 0.003275 65.5092541  6.6 mg/dL hApoB; gko f2 0.017319346.3860878 34.6 mg/dL Serum diluted 1:20,000

The apo(a) protein was present in serum of both male and female mousebefore puberty. Male mouse after puberty have significantly orcompletely repressed apo(a) protein expression due to elevatedtestosterone levels. Apo(a) expression in male mouse may be restored viacastration, continuous infusion of growth hormone via osmotic pump, orby biochemical modulation by dietary, chemical, or biological inducers.

Human apo B-100 protein expression: The presence of human apoB-100protein in serum was determined by ELISA in a serum drawn from the GULO(−/−) mouse, the apo(a); GULO(−/−) mouse, the apoB; GULO (−/−) mouse,and the Lp(a); GULO (−/−) mouse.

Presence of apoB-100 protein in mouse serum was determined by usingAssaypro (St. Charles, MO) AssayMax Human Apolipoprotein enzymeimmunoassay which is human apoB-100 specific and does not cross-reactwith mouse apoB-100, and/or with any other of the apolipoproteins (ApoAI, ApoC, ApoE).

Human apoB-100 was detected in the sera of hApoB-100; GULO (−/−) mouse,hApoB-100; apo(a); GULO(−/−) mouse, but not apo(a); GULO (−/−) mouse orGULO (−/−) mouse (Table 5).

Serum apo(a) protein was present in apo(a) gene containing GULO (−/−)mouse, apo(a) and human ApoB-100 gene containing GULO (−/−) mouse, butnot GULO (−/−) mouse without the transgene nor in GULO (−/−) mouse withonly human apo B-100 (Table 6)). These results confirm expression andtranslation of the human transgene apo(a) to serum protein apo(a).

TABLE 6 Female and male apo(a) expression. Male mouse may have lowexpression because of high testosterone levels in blood. Sample namesmg/dL apo(a) Comments GKO 0.66168 (below This test does not cross reactwith plasminogen or LDL. m1 detection limit) GKO 0 (below detectionlimit) m2 GKO f1 −0.33084 (below No apo(a) detected in background GULOKOmice. detection limit) GKO f2 −0.33084 (below No apo(a) detected inhApoB; GULOKO mice. detection limit) Lp(a); 0.16542 (below Extremelyhigh apo(a) detected in apo(a); GULOKO gko m1 detection female mice.limit) Lp(a); 0.16542 (below No apo(a) detected in apo(a); GULOKO malemice. gko m2 detection limit) Lp(a); 86.51466 (#464) Extremely highapo(a) detected in Lp(a); GULOKO gko f1 female mice, either generationF1 or F2. Lp(a); 79.4016 (#110) No apo(a) detected in Lp(a); GULOKO malemice, either gko f2 F1 or F2. apo(a); 0.33084 (below detection limit)gko m1 apo(a); −0.33084 (below Sex steroid testosterone suppressesapo(a) production in gko m2 detection these mice. limit) apo(a);133.8248 Orchidectomy may have to be performed in order to gko f1express Lp(a). apo(a); 156.1565 gko f2 hApoB; 0.16542 (below (#110 -ApoB gene signal = 13.5, apo(a) gene signal = gko m1 detection 3.7)limit) hApoB; 0 (below (#464 - ApoB gene signal = 20.4, apo(a) genesignal = gko m2 detection 8.5) limit) hApoB; −0.16542 (below (#456 -ApoB gene signal = 18.8, apo(a) gene signal = gko f1 detection 6.4)limit) hApob; 0.66168 (below detection limit) gko f2

Human apo (a) protein expression: Presence of apo (a) protein in serumwas determined by using the IBL International GmbH Lp(a) Enzymeimmunoassay which is human apo (a) specific and does not cross-reactwith plasminogen or LDL. All known isoforms of apo(a) can be detected.

Human Lp(a) protein expression: The Lp(a) particles are composed ofhuman apo(a) protein linked to human apo B-100 (the main protein of theLDL particle) by a disulfide bond.

SPIFE Cholesterol Profiling (FIG. 2): The presence of complete Lp(a)lipoprotein particles in the Lp(a)+GULO(−/−) transgenic mouse serum wasconfirmed using the electrophoresis method with Helena (Beaumont, Tex.)SPIFE Cholesterol Profiling and Immunofixation electrophoresis (IFE).

The Lp(a)-cholesterol band runs at a specific migration distance inrespect to LDL-cholesterol, and HDL-cholesterol, and it is found inhuman Lp(a)+; GULO(−/−) mouse sera, but not in the sera of GULO (−/−),human apo(a); GULO(−/−), or human apoB; GULO (−/−) mouse confirming thatthe presence of both human apoB-100 and human apo(a) are necessary toform complete Lp(a) particles in serum and that human apo(a) alone isinsufficient to produce Lp(a) and it does not link to mouse LDL viadisulfide bonds. In FIG. 2 the band closest to the top of the gelcorresponds to LDL-cholesterol, and the band furthest from the top toHDL-cholesterol. The tight, middle bands located between the LDL and HDLbands which are present in lanes 16-18 represent the Lp(a)-cholesterolfrom three different female Lp(a); GULO(−/−) mice. These bands aremissing from those GULO(−/−) mice not simultaneously expressing bothhuman apo(a) and human apoB-100 transgenes. Lane 19 representscharge-mass shifts resulting from a 24 hour room temperature incubationof serum specimen #18, which indicates that a small shift in theparticle migration may relate to lipoprotein oxidation.

Immunofixation Electrophoresis (IFE) using the mouse sera was conductedon individual apo(a) (FIG. 3) containing particles and human apoB-100containing particles (FIG. 4) as well, using human specific apo(a) andapoB-100 antibodies at Health Diagnostic Laboratory, Inc. (Richmond,Va.). The bands represent apo(a) and apoB-100 protein respectively intransgenic mouse and visualization of the same in serum derived fromfemale and male mouse.

Confirming a lack of vitamin C production in the transgenic mousestrains: Serum level of ascorbate (vitamin C) in both GULO(−/−) mouseand a newly generated strain of Lp(a)+GULO (−/−) depends on its dietarysupplementation. Mouse kept on vitamin C deficient diet has a graduallydiminishing serum concentration of vitamin C until it reaches zero orthe mouse dies. Serum levels of vitamin C were obtained using theBiovision (Mountain View, Calif.) Ferric Reducing Ascorbate Assay(FRASC) Kit (FIG. 5).

Lipoprotein-cholesterol, Apo(a) particle, and ApoB-100 particleModulation (FIG. 6, FIG. 7, and FIG. 8): The analysis was conducted onmouse sera from the Lpa+Gulo (−/−)mouse supplemented with either 30mg/L, 60 mg/L or 150 mg/L of ascorbic acid provided in drinking water inaddition to 500 ppm vitamin C provided in food (full supplementation).It was observed that the whole spectrum of lipoprotein cholesterolsand/or lipoprotein particles could be modulated by dietary ascorbatealone. These data give additional particle number data and inconjunction with the lipoprotein cholesterol load data providedcomprehensive confirmation of the presence of apo(a) protein, humanapoB-100 protein, and disulfide linked Lp(a) in these mouse sera.

Sample order in FIGS. 6-8 correspond to the following key, with 30 vcreferring to 30 mg/L Vitamin C group, 60 vc referring to 60 mg/L VitaminC group, and sc referring to fully supplemented (150 mg/L Vitamin C+500ppm Vitamin C in food) control group. The first three wells of each rowwere not used.

TABLE 7 Sample list for the wells in FIG. 6, 7 and 8. Well# Group SampleID 4 30vc1f e710f 5 30vc2f e689f 6 30vc2f e598f 7 30vc2f e704f 8 30vc2fe723f 9 30vc2f e717f 10 human control 11 30vc1f fell off 1 12 30vc1fe762f 13 30vc1f e759f 14 30vc1f e699f 15 30vc1f e706f 16 30vc1f fell off2 17 60vc2f e773f 24 60vc2f ea315f 25 60vc2f e771f 26 60vc2f e694f 2760vc2f e761f 28 60vc2f e768f 29 60vc1f e776f 30 human control 31 60vc1fe777f 32 60vc1f e780f 33 60vc1f fell off 34 60vc1f e733f 35 60vc1f e784f36 sc2f e741f 37 sc2f e814f 44 sc2f fell off 2 45 sc2f fell off 1 46sc2f e781f 47 sc1f e732f 48 sc1f e730f 49 sc1f e739f 50 human control 51sc1f e731f 52 sc1f e812f 53 sc1f e832f 54 sc2f e778f 64 30vc2m e697m 6530vc2m e698m 66 30vc2m e770m 67 30vc2m fell off 68 30vc2m e695m 6930vc1m e683m 70 human control 71 30vc1m e684m 72 30vc1m fell off 7330vc1m e693m 74 30vc1m e727m 75 60vc2m e782m 76 60vc2m fell off 2 7760vc2m fell off 1 78 60vc2m e809m 79 60vc2m e738m 84 60vc1m e811m 8560vc1m e736m 86 60vc1m fell off 87 60vc1m e734m 88 60vc1m e740m 89 sc2mfell off 2 90 human control 91 sc2m e724m 92 sc2m e605m 93 sc2m e702m 94sc2m fell off 1 95 sc1m e705m 96 sc1m e718m 97 sc1m e701m 98 sc1m e703m99 sc1m e596m

INDUSTRIAL APPLICATION

Crossbreeding a dual transgenic mouse to produce a human Lp(a) and notproduce vitamin C due to lack of GULO (GULO−/−) gene using transgenicmouse having a first knockout strain, a second strain to make a thirdstrain and using the first knockout strain and fourth strain to make afifth strain, using the third strain and the fifth strain to make a dualtransgenic mouse. Treating the dual transgenic mouse with aLp(a)-modulating compounds in order to identify preventive and/ortherapeutic approaches for a human Lp(a)-related diseases. The humanLp(a)-related disease is cardiovascular, inflammatory, infectious ordegenerative in nature.

What is claimed is:
 1. A dual transgenic mammal, comprising: a thirdstrain of transgenic mammal was obtained by crossbreeding a firstknockout strain mammal and a second strain of transgenic mammalexpressing a set of genes that are human in nature, wherein the firstknockout strain mammal is lacking a gulonolactone oxidase gene, GULOgene (GULO−/−), and produces no Vitamin C, wherein the second strain ofmammal possesses a human apolipoprotein (a) gene (apo (a)+), wherein thethird strain of transgenic mammal expresses the human apo (a) gene (apo(a)+) and, simultaneously lacks the GULO gene (GULO−/−); and a fifthstrain of mammal made by crossbreeding the first knockout strain mammallacking the GULO gene and the fourth strain of mammal possessing a humanapolipoprotein B-100 gene (apo B-100+), wherein the fifth strain ofmammal lacks the GULO gene (GULO−/−) and expresses the human apo B-100gene (apo B-100+).
 2. The transgenic mammal of claim 1, furthercomprising: a dual transgenic mammal that lacks the GULO gene (GULO−/−)and, simultaneously, produces human lipoprotein (a) (Lp(a)+) wasobtained by crossbreeding the third strain of mammal lacking GULO gene(GULO−/−) and, simultaneously expressing the human apo (a) gene (apo(a)+), and the fifth strain of transgenic mammal lacking the GULO gene(GULO−/−) and, simultaneously, possessing the human apo B-100 gene (apo(B-100)+).
 3. The transgenic mammal of claim 3, wherein the firstknockout mammal, second, third, fourth, fifth transgenic mammal and thedual transgenic mammal is a mouse.
 4. A method, comprising:crossbreeding a first knockout strain and a second strain of mouse toobtain and a third strain of transgenic mouse that do not producevitamin C and expresses a first human gene to produce the humanproducts; crossbreeding the first knockout strain and a third strain ofmouse to produce a fifth transgenic mouse to express a second human geneand produce no vitamin C; and crossbreeding the fifth strain oftransgenic mouse and the third strain of transgenic mouse to producenovel dual transgenic mouse that expresses the first human gene, thesecond human gene and a third human gene to make a first product, secondproduct and a third product but produce no vitamin C.
 5. The method ofclaim 4, further comprising: testing the transgenic mouse using aspecific sequence of primer to confirm the presence of the two humangenes, human apo (a) and human apo B-100, the first product, secondproduct, third product and lack of GULO gene.
 6. The method of claim 4,wherein the first strain of transgenic mouse lacks the GULO gene(GULO−/−), the second strain of transgenic mouse expresses human apo (a)(+), the third strain lacks the GULO gene (GULO−/−) and expresses humanapo (a) (apo (a)+), the fourth strain of transgenic mouse expresseshuman apo B-100 (B-100+), the fifth strain lacks the GULO gene (GULO−/−)and, simultaneously, expresses human apo B-100 (B-100+).
 7. The methodof claim 5, wherein the specific sequences are Seq 1 to Seq
 12. 8. Themethod of claim 4, further comprising: crossbreeding the third strainexpressing human GULO (GULO−/−) and human (apo (a)+), and the fifthstrain of transgenic mouse expressing human GULO (GULO−/−) and human apoB-100(B-100+) to obtain a dual transgenic mouse.
 9. The method of claim9, wherein the dual transgenic mouse lack the ability to express thegulonolactone oxidase gene [GULO (GULO−/−)] and produces humanlipoprotein (a) (Lp(a)).
 10. The method of claim 6, wherein the firstproduct is human apo (a), second product is human apo B-100 and thethird product is human Lp(a).
 11. The method of claim 9, furthercomprising: testing an effect of a pharmaceutical drug that will affectLp(a) synthesis and/or Lp(a) plasma levels and/or Lp(a) depositioninside the vascular wall in the transgenic mouse with the purpose totreat a cardiovascular disease.
 12. A method, comprising: crossbreedinga dual transgenic mouse to produce human Lp(a) and not produce Vitamin Cdue to lack of GULO (GULO−/−) gene using transgenic mouse having a firstknockout strain, a second strain to make a third strain and using thefirst knockout strain and fourth strain to make a fifth strain, usingthe third strain and the fifth strain to make a dual transgenic mouse;and treating the dual transgenic mouse with Lp(a)-modulating compoundsin order to identify preventive and/or therapeutic approaches for ahuman Lp(a)-related diseases.
 13. The method of claim 12, wherein thefirst knockout strain of transgenic mouse lacking the GULO gene(GULO−/−), the second strain of transgenic mouse expresses human apo (a)(apo(a)+), the third strain lacks the GULO gene (GULO−/−) and,simultaneously, possesses human apo(a) (apo (a)+) gene, the fourthstrain of transgenic mouse possesses human apo B-100 (B-100+), the fifthstrain lacks the GULO gene (GULO−/−) and, simultaneously, expresseshuman apo B-100 (B-100+).
 14. The method of claim 13, wherein the firstknockout strain does not produce Vitamin C.
 15. The method of claim 13,wherein the second strain of transgenic mouse produces human apo (a).16. The method of claim 13, wherein the third strain of transgenic micedoes not produce Vitamin C and produces human apo (a).
 17. The method ofclaim 13, wherein the fifth strain of transgenic mouse does not produceVitamin C and produces human apo B-100.
 18. The method of claim 12,wherein the dual transgenic mouse lacks the GULO gene and does notproduce Vitamin C and, simultaneously produces human Lp(a).
 19. Themethod of claim 12, wherein the human Lp(a)-related disease is acardiovascular disease.
 20. The method of claim 12, wherein the humanLp(a)-related disease is inflammatory, infectious or degenerative innature.